WO2011062298A1 - Système pour accroître l'expression génétique, et vecteur de support pour un tel système - Google Patents

Système pour accroître l'expression génétique, et vecteur de support pour un tel système Download PDF

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WO2011062298A1
WO2011062298A1 PCT/JP2010/071196 JP2010071196W WO2011062298A1 WO 2011062298 A1 WO2011062298 A1 WO 2011062298A1 JP 2010071196 W JP2010071196 W JP 2010071196W WO 2011062298 A1 WO2011062298 A1 WO 2011062298A1
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gene
promoter
expression
construct
enhancer
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PCT/JP2010/071196
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English (en)
Japanese (ja)
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公文裕巳
許南浩
阪口政清
渡部昌実
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国立大学法人岡山大学
桃太郎源株式会社
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Priority to US13/510,719 priority Critical patent/US9493776B2/en
Priority to JP2011541992A priority patent/JP5697042B2/ja
Priority to AU2010322723A priority patent/AU2010322723B9/en
Priority to CA2781332A priority patent/CA2781332C/fr
Priority to EP20100831684 priority patent/EP2508603B1/fr
Priority to CN201080061897.9A priority patent/CN102741405B/zh
Priority to KR1020127015275A priority patent/KR101752941B1/ko
Priority to RU2012125253/10A priority patent/RU2577971C2/ru
Publication of WO2011062298A1 publication Critical patent/WO2011062298A1/fr
Priority to IL219882A priority patent/IL219882A/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the present invention relates to a method for increasing gene expression using a promoter, an enhancer and the like, and an expression cassette for increasing gene expression including the promoter, enhancer and the like.
  • Patent Documents 1 to 4 various gene expression promoters such as CMV promoter and CAG promoter have been developed to increase gene expression efficiency.
  • CMV promoter and CAG promoter have been developed to increase gene expression efficiency.
  • problems such as almost no gene expression or a very small amount of expressed protein occur on a daily basis, depending on the cell type and gene type. .
  • This problem is also a major barrier in the development of medicine using gene expression for diagnosis and treatment.
  • the present inventor attempted to develop a new system capable of expressing a gene with higher efficiency in a gene expression system using a promoter, and compared and examined promoter activities by a combination of promoters and enhancers of various genes. It was. As a result, the gene to be expressed downstream of the first promoter and the DNA construct containing the poly A addition sequence are expressed using a gene expression cassette in which an enhancer or a second promoter is connected downstream of the DNA construct.
  • a gene expression cassette comprising a DNA construct comprising a gene to be expressed downstream of a first promoter and a poly A addition sequence, and further comprising an enhancer or a second promoter downstream of the DNA construct.
  • the enhancer is at least one enhancer selected from the group consisting of a CMV enhancer, an SV40 enhancer, and an hTERT enhancer.
  • the expression cassette of [8], wherein the tumor suppressor gene is a REIC / Dkk-3 gene.
  • the expression cassette according to [9], wherein the gene to be expressed is a fragment DNA of the REIC / Dkk-3 gene.
  • the cassette for expression of the foreign gene of [1] which has a structure shown in FIG. 21 (FIG. 58).
  • [14] A vector comprising a cassette for expressing a foreign gene according to any one of [1] to [13].
  • [16] A host cell comprising the vector of [14] or [15].
  • [17] A disease detection or treatment preparation comprising the vector of [14] or [15].
  • a DNA construct containing a gene to be expressed and a poly A addition sequence downstream of the first promoter and an enhancer or second promoter linked to the DNA construct are introduced into a vector, and the vector is used. To express the gene.
  • An expression cassette comprising introducing the expression cassette according to any one of [1] to [13] or the vector according to [14] or [15] into a cell and culturing the cell.
  • FIG. 1 shows the expression of various foreign genes transfected into HEK293 cell line using FuGENE TM -HD for 36 hours.
  • FIG. 2 shows the expression of KLF gene transfected into various cell lines using FuGENE TM -HD for 36 hours.
  • FIG. 3 shows the expression of KLF gene transfected into HEK293 cell line for 36 hours using various transfection reagents.
  • FIG. 4 is a diagram showing the expression when the HEK293 cell line was transfected with a gene encoding full-length REIC gene and N78-REIC for 36 hours using FuGENE TM -HD.
  • FIG. 5A shows a construct No. in which DNA encoding N78-REIC was inserted.
  • FIG. 14 is a graph (graph) showing growth suppression and cell death induction of human prostate cancer cell PC3 cell line according to FIG. FIG. 5-2 shows construct No. 5 inserted with DNA encoding N78-REIC.
  • FIG. 14 is a diagram (photograph) showing growth suppression and cell death induction of human prostate cancer cell PC3 cell line according to FIG. FIG. 6 shows construct No. in which the full-length REIC gene was inserted.
  • FIG. 14 shows growth inhibition of human prostate cancer cell PC3 cell line according to FIG. FIG. FIG. FIG. It is a figure which shows the structure of 2.
  • FIG. FIG. FIG. FIG. FIG. FIG. 6 is a diagram showing the structure of FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. 18 shows the structure No.
  • FIG. 21 is a diagram showing the structure of an adenovirus construct encoding the full-length REIC / Dkk-3 gene.
  • FIG. 22-1 is a diagram showing the entire base sequence of pDNR-1r Donor vector.
  • FIG. 22-2 is a diagram showing the entire base sequence of pDNR-1r Donor vector (continuation of FIG. 22-1).
  • FIG. 23 is a diagram showing the entire base sequence of the pIDT-SMART vector.
  • FIG. 24 is a diagram showing the base sequence of the CMV i promoter (hCMV + intron promoter) region.
  • FIG. 25 is a diagram showing the base sequence of the BGH polyA (3 ⁇ stop + BGH polyA) region.
  • FIG. 26 is a diagram showing the base sequence of the CMV enhancer region.
  • FIG. 27 shows the nucleotide sequence of the human ⁇ -actin promoter region.
  • FIG. 28 is a diagram showing the base sequence of the RU5 ′ forward direction ⁇ HTLV Type 1 long terminal repeat R segment and part of the U5 sequence (R-U5 ′) ⁇ region.
  • FIG. 29 is a diagram showing the base sequence of the RU5 ′ reverse direction ⁇ HTLV Type 1 long terminal repeat R segment and part of the U5 sequence (R-U5 ′) ⁇ region.
  • FIG. 30 is a diagram showing the base sequence of the 4 ⁇ CMV enhancer region.
  • FIG. 31 shows the base sequence of the CAG promoter region.
  • FIG. 32 shows the base sequence of the 2IRES insert region.
  • FIG. 33 shows the nucleotide sequence of the SV40ori-UAS-CMVi-RU5 ′ region.
  • FIG. 34 is a diagram showing the base sequence of 3 ⁇ stop-BGH-polyA-UAS-hTERT enhancer + SV40 enhancer + CMV enhancer region.
  • FIG. It is a figure which shows the whole base sequence of 14 vectors.
  • FIG. It is a figure which shows the whole base sequence of 14 vectors (continuation of FIG. 35-1).
  • FIG. 36 shows the structure No. FIG. FIG. FIG. FIG.
  • FIG. 38 is a diagram showing the nucleotide sequence of the SV40ori-UAS-SV40 enh-intron A-RU 5 ′ region. It is a figure which shows the base sequence of the insertion part of the left side (upstream side) from 15 insertion genes (target gene).
  • FIG. 39 is a diagram showing the base sequence of the SV40ori-UAS-hTERT enh-intron A-RU 5 ′ region. It is a figure which shows the base sequence of the insertion part of the left side (upstream side) from 16 insertion genes (target gene).
  • FIG. 40 shows the nucleotide sequence of the GFP region in the plasmid.
  • FIG. 40 shows the nucleotide sequence of the GFP region in the plasmid.
  • FIG. 41-2 shows the structure No. It is a figure which shows the base sequence of the plasmid which inserted DNA of the GFP gene in 14 insertion gene (target gene) area
  • FIG. 42-1 shows the structure No. It is a figure which shows the base sequence of the plasmid which inserted DNA of the GFP gene in 15 insertion gene (target gene) area
  • FIG. 42-2 shows construct no. It is a figure which shows the base sequence of the plasmid which inserted DNA of the GFP gene in 15 insertion gene (target gene) area
  • FIG. 44 is a diagram showing the intensity of GFP gene expression when a plasmid containing the SV40 promoter (construct No. 15) and the hTERT promoter (construct No. 16) is used.
  • FIG. 45 shows the nucleotide sequence of the human erythropoietin region in the plasmid.
  • FIG. 47A is a view showing a base sequence of a human IgG light chain region (FIG. 44A) in a plasmid.
  • FIG. 47B is a diagram showing a base sequence of a human IgG heavy chain region (FIG. 44B) in a plasmid.
  • FIG. It is a figure which shows the result at the time of expressing human IgG light chain (FIG. 48A) and human IgG heavy chain (FIG. 48B) using 14 plasmids.
  • FIG. FIG. 50 shows construct NO.
  • FIG. 52 is a diagram showing the sequence of the c-myc gene.
  • FIG. 53 shows construct no. It is a figure which shows the base sequence which BGH polyA and three enhancers which exist downstream of the expression gene of 14 expression plasmids connected.
  • FIG. 54 shows construct No.
  • FIG. 59-1 shows the structure No. It is a figure which shows the base sequence of the plasmid which inserted DNA of the GFP gene in 21 insertion gene (target gene) area
  • 59-2 shows the structure No.
  • FIG. 59 is a diagram showing the base sequence of a plasmid in which GFP gene DNA is inserted into 21 inserted gene (target gene) regions (continuation of FIG. 59-1).
  • FIG. 60 shows the results when the target gene is inserted into a plasmid containing the hTERT promoter and expressed.
  • FIG. 61 is a diagram showing expression when a plurality of expression vectors incorporating different foreign genes are co-transfected.
  • FIG. 62 shows the production of human erythropoietin using the expression vector of the present invention.
  • FIG. 63 is a diagram showing the purity of human erythropoietin produced using the expression vector of the present invention.
  • FIG. 64 is a view showing the production amount of human erythropoietin using the expression vector of the present invention, FIG. 64A shows the production amount in 25 mL of culture supernatant, and FIG. 64B shows the equivalent production amount in 1 L of culture supernatant.
  • FIG. 65 is a diagram showing production of human REIC protein using the expression vector of the present invention.
  • FIG. 66 is a diagram showing an ion exchange column chromatogram of human REIC protein produced using the expression vector of the present invention.
  • FIG. 67 is a diagram showing the production amount of human REIC protein (the production amount in 520 mL of culture supernatant and the equivalent production amount in 1 L of culture supernatant) using the expression vector of the present invention.
  • the expression cassette for the protein to be expressed refers to a set of DNAs that enable the expression of the protein to be expressed.
  • the expression cassette includes a DNA construct containing a gene of a protein to be expressed (gene to be expressed) and a poly A addition sequence at least downstream of the first promoter, and further an enhancer downstream of the construct. Or it has the structure where the 2nd promoter is connected and contained. Any gene can be used for expression.
  • the site into which the gene to be expressed is inserted may exist as a multicloning site. In this case, the gene to be expressed may be inserted into the multicloning site (insertion site) using a sequence recognized by a restriction enzyme.
  • the expression cassette according to the present invention also includes an expression cassette that does not contain the gene DNA itself to be expressed as described above and that includes a portion into which the DNA is inserted as a multicloning site.
  • a gene to be expressed may be called a target gene or target gene, and a protein may be called a target protein or target protein. From the viewpoint of constructing an expression cassette, these genes are also referred to as inserted genes because they are inserted into the target gene region of the expression cassette. It may also be a foreign gene. Further, the enhancer or the second promoter is present at the most downstream of the expression cassette of the present invention, and there is no other gene expression mechanism downstream thereof.
  • the expression cassette of the present invention has a structure in which at least a gene to be expressed is sandwiched between one first promoter and at least one enhancer, or between one first promoter and one second promoter.
  • the other gene expression mechanism means a mechanism for expressing a gene other than the gene to be expressed, and a promoter or enhancer for expressing a gene other than the gene to be expressed. Etc. are included.
  • promoters may exist upstream and downstream of the gene to be expressed, but these two promoters are used for enhancing the expression efficiency of the gene to be expressed.
  • the expression cassette of the present invention is incorporated into an expression vector.
  • the present invention also includes a vector comprising the expression cassette of the present invention.
  • the enhancer or the second promoter is present in the most downstream of the expression cassette of the present invention, and there is no other gene expression mechanism downstream thereof.
  • the vector containing the expression cassette of the present invention has no other gene expression mechanism downstream of the expression cassette.
  • the gene to be expressed includes a gene (DNA) encoding a foreign protein that has been artificially inserted, and includes genes that have different origins from host cells and genes that have the same origin.
  • the foreign gene is also referred to as an inserted gene.
  • the type of gene to be expressed is not limited.
  • REIC / Dkk-3 gene (SEQ ID NO: 17 shows the nucleotide sequence)
  • tumor suppressor genes such as p53 and Rb, interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL- 14, IL-15, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, angiostatin, thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, tumor necrosis Factors, hepatocyte growth factor, erythropoietin,
  • Proteins which can be used as medicaments, such as physiologically active substances, experiments in diagnostics and laboratories for use in the detection of such diseases include genes encoding proteins useful as a reagent for use in research or the like.
  • the target protein to be expressed is preferably an extracellular secreted protein.
  • the expression of the gene inherent in the organism can be enhanced by artificially linking an enhancer or promoter downstream of the gene inherent in the organism. That is, the gene to be expressed in the present invention includes a gene originally possessed by an organism.
  • the present invention also includes inserting an enhancer or promoter downstream of the gene inherent in the organism and controlling the expression of the gene.
  • the cell which inserted the enhancer or the promoter downstream of this gene of the cell containing the gene which an organism originally has is also included.
  • genes to be expressed include DNA encoding siRNA, shRNA, miRNA and the like having RNA interference action.
  • RNA having RNA interference action is used, expression of a specific gene can be suppressed by transcription-producing these RNAs using a transfer RNA promoter.
  • the REIC / Dkk-3 gene and the like can be used for tumor treatment, and not only the full-length gene but also a fragment thereof can be used.
  • REIC / Dkk-3 shown in SEQ ID NO: 18 An amino acid sequence beginning with the first amino acid in the amino acid sequence of the protein and ending with any of the 39th to 78th amino acids, or the amino acid sequence of the REIC / Dkk-3 protein shown in SEQ ID NO: 18; It consists of an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence starting with the first amino acid and ending with any of the 39th amino acid to the 78th amino acid, and has an apoptotic activity DNA encoding a polypeptide having Among these DNAs, a DNA (N78-REIC DNA) encoding a peptide consisting of amino acids 1 to 78 of the amino acid sequence of the REIC / Dkk-3 protein shown in SEQ ID NO: 18 is preferably used.
  • a reporter gene may be included for detection or diagnosis of a disease.
  • a promoter is a specific base sequence on DNA that initiates transcription using DNA as a template, and generally has a common sequence.
  • prokaryotes such as Escherichia coli usually have a TATAATG sequence at the ⁇ 10 base pair site of the transcription initiation site and a TTGACA sequence at the ⁇ 35 base pair site.
  • eukaryotes usually have a TATA box at a -20 base pair site.
  • the expression cassette of the present invention may necessarily have a first promoter upstream of the gene to be expressed, and may have a second promoter downstream of the gene to be expressed.
  • the promoters used as the first promoter and the second promoter are not limited, and the first promoter and the second promoter may be the same or different.
  • a non-specific promoter capable of promoting the expression of a foreign gene in any cell or tissue and a specific or selective promoter such as a tissue or organ-specific promoter, a tumor-specific promoter, a developmental or differentiation-specific promoter, etc. can be used.
  • a specific promoter can be used as the first promoter
  • a non-specific promoter can be used as the second promoter.
  • hTERT human telomerase reverse transcriptase
  • PSA prostate specific antigen
  • c-myc GLUT promoter and the like
  • OCT3 / 4, NANOG promoter, etc. neural stem cell-specific promoters such as Nestin promoter, cell stress sensing promoters such as HSP70, HSP90, p53 promoter, hepatocyte-specific promoters such as albumin promoter, etc.
  • Proliferative cell-specific promoters include TNF-alpha promoters as susceptibility promoters and SV40 promoters as promoters that increase the number of copies of infectious plasmids.
  • CMV-i promoter hCMV + intron promoter
  • ⁇ -actin promoter ⁇ -actin promoter
  • CAG promoter SEQ ID NO: 12
  • CMV promoter and the like are used as the first promoter.
  • a CMV promoter or the like is used as the promoter of the above.
  • the animal species from which the ⁇ -actin promoter is derived is not limited, and a mammalian ⁇ -actin promoter such as a human ⁇ -actin promoter (SEQ ID NO: 8) or a chicken triactin promoter is used.
  • Artificial hybrid promoters such as the CMV-i promoter described above can also be used.
  • the CMV-i promoter can be synthesized based on the description in US Pat. No. 5,168,062 and US Pat. No. 5,385,839.
  • a core promoter portion consisting of a minimal sequence having promoter activity may be used.
  • the core promoter refers to a promoter region having a function of inducing accurate transcription initiation, and may include a TATA box.
  • cancer / tumor-specific promoters such as the hTERT promoter can be suitably used for gene therapy by gene expression and cancer diagnosis targeting cancer. 21 can be used for such applications.
  • polyadenylation sequence is not limited, and is included in the growth hormone gene-derived poly A addition sequence, for example, the bovine growth hormone gene-derived poly A addition sequence (included in the base sequence shown in SEQ ID NO: 6). (Sequence after the 13th base)), poly A addition sequence derived from human growth hormone gene, poly A addition sequence derived from SV40 virus, poly A addition sequence derived from human or rabbit ⁇ -globin gene, and the like. Inclusion of a poly A addition sequence in an expression cassette increases transcription efficiency.
  • the enhancer is not limited as long as it enhances the amount of messenger RNA (mRNA) produced by transcription.
  • An enhancer is a base sequence that has the effect of promoting the action of a promoter, and generally there are many that consist of around 100 bp. Enhancers can promote transcription regardless of sequence orientation.
  • One type of enhancer may be used in the present invention, but a plurality of two or more identical enhancers may be used, or a plurality of different enhancers may be used in combination.
  • the order is not limited.
  • a CMV enhancer SEQ ID NO: 7
  • SV40 enhancer SV40 enhancer
  • hTERT Telomerase Reverse transcriptase
  • a hTERT enhancer, an SV40 enhancer and a CMV enhancer connected in this order can be mentioned.
  • a plurality of enhancers for example, 1 to 4 enhancers, may be linked upstream of the DNA construct containing the DNA encoding the protein to be expressed and the poly A addition sequence.
  • the enhancer linked upstream is not limited, but a CMV enhancer is preferable.
  • 4 ⁇ CMV enhancer (SEQ ID NO: 11) in which four CMV enhancers are linked can be used.
  • the combination of the CMV i promoter and the CMV enhancer allows the strong expression of the gene to be expressed when any gene is inserted in almost all cells (host cells), regardless of the transfection reagent used. Protein expression is possible.
  • a specific cell for example, HEK293 cell line or MCF7 cell line in the examples described later
  • a specific gene for example, REIC / Dkk in the examples described later. Expression is further enhanced in the -3 gene and CD133 gene.
  • the expression is further enhanced depending on specific cells (for example, HepG2 cell line and HeLa cell line in Examples described later).
  • RU5 ′ (SEQ ID NO: 9) may be linked immediately upstream of the DNA encoding the protein to be expressed.
  • the term “immediately upstream” means that they are directly linked without intervening elements having other specific functions, but a short sequence may be included as a linker.
  • RU5 ′ is an HTLV-derived LTR, and is an element that increases protein expression by insertion (Mol. Cell. Biol., Vol. 8 (1), p. 466-472, 1988).
  • UAS may be linked immediately upstream of the enhancer and / or promoter.
  • UAS is a binding region of the GAL4 gene, and protein expression is increased by inserting the GAL4 gene later.
  • SV40-ori may be connected to the uppermost stream of the expression cassette. SV40-ori is a binding region of the SV40 gene, and protein expression increases by inserting the SV40 gene later.
  • the term “functionally linked” means that each element is linked so as to enhance the expression of the gene to be expressed by exerting its function.
  • constructs are also referred to as a plasmid backbone.
  • construct No. 2 (FIG. 8), no. 4 (FIG. 10), no. 6 (FIG. 12), no. 8 (FIG. 14), no. 10 (FIG. 16), no. 12 (FIG. 18), no. 14 (FIG. 20), no. 15 (FIG. 36), no. 16 (FIG. 37), no. 17 (FIG. 49), no. 20 (FIG. 57) and no. 21 (FIG. 58).
  • construct shown in FIG. 8 construct No. 2 (FIG. 8), no. 4 (FIG. 10), no. 6 (FIG. 12), no. 8 (FIG. 14), no. 10 (FIG. 16), no. 12 (FIG. 18), no. 14 (FIG. 20), no. 15 (FIG. 36), no. 16 (FIG. 37), no. 17 (FIG. 49), no. 20 (FIG. 57) and no. 21 (FIG. 58).
  • construct No. 3 in which three enhancers are linked downstream of the target gene. 14, construct no. 15 and construct no. Sixteen constructs are the best.
  • construct no. By using the REIC full-length gene as the target gene in 14, the protein expression level is comparable to that in the case of an adenovirus vector (encoding the full-length REIC / Dkk-3 gene) at 100 MOI.
  • the construction No. The plasmid backbone of 14 shows an effect of increasing gene expression even in a gene fragment such as DNA encoding N78-REIC. For example, construct no.
  • the DNA represented by the nucleotide sequence of SEQ ID NO: has a mutation in the nucleotide sequence as long as it has the activity of each DNA or the activity of the protein or polypeptide encoded by each DNA. It may be.
  • BLAST Basic Local Alignment
  • DNA encoding a protein or polypeptide consisting of can also be used for the construction of the DNA construct of the present invention.
  • stringent conditions are, for example, conditions of about “1XSSC, 0.1% SDS, 37 ° C.”, and more severe conditions are “0.5XSSC, 0.1% SDS, 42 ° C.” The condition is about “0.2XSSC, 0.1% SDS, 65 ° C.”.
  • isolation of DNA having high homology with the probe sequence can be expected as the hybridization conditions become more severe.
  • the above combinations of SSC, SDS, and temperature conditions are examples, and necessary stringency can be realized by appropriately combining the DNA concentration, DNA length, hybridization reaction time, and the like. is there.
  • vectors for inserting the expression cassette of the present invention include plasmids, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, herpes virus vectors, Sendai virus vectors, and other viral vectors and biodegradable polymers.
  • Non-viral vectors can be mentioned. What is necessary is just to introduce
  • a target gene By introducing a vector into which a cassette for expression of the present invention is inserted into a cell and transfecting the cell, a target gene can be expressed in the cell to produce a target protein.
  • a eukaryotic cell or a prokaryotic cell system can be used.
  • eukaryotic cells include cells such as established mammalian cell lines, insect cell lines, filamentous fungal cells, and yeast cells.
  • prokaryotic cells include E. coli, Bacillus subtilis, and Brevibacillus bacteria. Bacterial cells are mentioned.
  • mammalian cells such as Hela cells, HEK193 cells, CHO cells such as CHO, COS cells, BHK cells, Vero cells are used.
  • the transformed host cell can be cultured in vitro or in vivo to produce the target protein.
  • Host cells are cultured according to a known method.
  • a known culture medium such as DMEM, MEM, RPMI 1640, and IMDM can be used as the culture solution.
  • the expressed protein can be purified by a known method from a culture solution in the case of a secreted protein or from a cell extract in the case of a non-secreted protein.
  • a cell When expressing and producing a target protein, a cell may be produced by simultaneously transfecting a plurality of vectors containing different target genes. By doing so, a plurality of proteins can be produced at one time.
  • a commercially available vector may be modified to include the expression cassette of the present invention.
  • an enhancer can be incorporated into the downstream region of the expression gene cassette of a commercially available vector such as a pShuttle vector.
  • the vectors of the present invention include those obtained by modifying commercially available cassettes.
  • the present invention further includes an adenovirus (Ad) vector and an adeno-associated virus (AAV) vector containing the above-described cassette for gene expression to be expressed.
  • Ad adenovirus
  • AAV adeno-associated virus
  • the vector can be prepared by inserting the above-described gene expression cassette into adenovirus or adeno-associated virus.
  • the characteristics of adenovirus vectors are (1) gene transfer into many types of cells, (2) gene transfer into cells in growth arrested phase efficiently, and (3) concentration by centrifugation. High titer (10 to 11 PFU / ml or more) virus can be obtained, and (4) suitable for direct gene transfer into tissue cells in vivo.
  • adenovirus for gene therapy a first generation adenovirus vector lacking the E1 / E3 region (Miyake, S., et. al. , Proc. Natl. Acad. Sci. USA.
  • a second generation adenoviral vector (Lieber, A., et. Al.) Lacking the E2 or E4 region in addition to the E1 / E3 region. al. , J .; Virol. , 70, 8944, 1996; Mizuguchi, H .; & Kay, M .; A. , Hum. Gene Ther. , 10, 2013, 1999), the adenoviral genome almost completely deleted (GUTESS), a third generation adenoviral vector (Steinwaderer, DS, et al., J.
  • Adeno-associated virus is a parvovirus genus single-stranded DNA virus, and has the characteristics of (1) gene long-term expression, (2) low toxicity, and (3) gene transfer into dividing and non-dividing cells. The formation of concatamers (complexes in which single-stranded DNAs are linked) is the cause of long-term gene expression.
  • the adenovirus (Ad) vector and the adeno-associated virus (AAV) vector that contain the cassette for expression of the gene to be expressed of the present invention and can express the target gene to be expressed are used for disease detection or treatment. Can be used.
  • Diseases include cancer and the like, and the cells are directed by a peptide such as RGD that selectively binds to a protein expressed in a specific cell inserted into the outer shell of an adenovirus vector or an adeno-associated virus vector.
  • a gene that infects and expresses the cells is expressed.
  • the gene to be expressed is a reporter gene such as a luciferase gene
  • specific cells can be detected by luminescence or the like.
  • a specific cell is a cancer cell, by administering to a subject, the cancer cell in a subject can be detected and it can be diagnosed that the subject is suffering from cancer.
  • the adenovirus vector and adeno-associated virus vector of the present invention can detect one cell, they can be used, for example, for detection of microcancer.
  • the gene to be expressed is a therapeutic gene
  • the target gene to be expressed in a specific cell is expressed, and a therapeutic effect can be exhibited.
  • a specific cell is a cancer cell and a cancer suppressor gene such as REIC / Dkk-3 gene is used
  • the gene for cancer treatment is delivered to the cancer cell of the subject by administration to the subject, and the cancer Genes are expressed in cells and exert therapeutic effects.
  • the present invention includes a diagnostic or therapeutic viral preparation comprising such an adenoviral vector or an adeno-associated viral vector.
  • examples of the cancer to be treated according to the present invention include brain / neural tumors, skin cancers, and the like.
  • Examples include cancer, testicular cancer, bone / osteosarcoma, leiomyoma, rhabdomyosarcoma, and mesothelioma.
  • the adenovirus vector and adeno-associated virus vector of the present invention can be used for the treatment of primary cancer and metastatic cancer.
  • the adenovirus vector and adeno-associated virus vector of the present invention can be used in the gene therapy field, for example, intravascular administration such as intravenous administration and intraarterial administration, oral administration, intraperitoneal administration, intratracheal administration, bronchial administration, and the like. It can be administered by internal administration, subcutaneous administration, transdermal administration, or the like.
  • the adenovirus vector and adeno-associated virus vector of the present invention are highly directed to specific tissues and cells, and can efficiently deliver target genes to specific tissues and cells. Efficient diagnosis and treatment.
  • the adenovirus vector and adeno-associated virus vector may be administered in a therapeutically effective amount.
  • a therapeutically effective amount can be readily determined by one skilled in the art of gene therapy.
  • the dose can be appropriately changed depending on the severity of the disease state, sex, age, weight, habits, etc. of the subject.
  • adenoviral vector and adeno-associated viral vector 11 ⁇ 2.0 ⁇ 10 12 virtual genome / kg body weight preferably 1.0 ⁇ 10 11 ⁇ 1.0 ⁇ 10 12 virtual genome / kg body weight, more preferably 1.0 ⁇ 10 11 ⁇ 5.0 ⁇ 10 11
  • What is necessary is just to administer in the quantity of a viral genome / kg body weight.
  • the virtual genome indicates the number of molecules (number of virus particles) of the genome of adenovirus or adeno-associated virus, and is sometimes referred to as particle.
  • aqueous solution for injection isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants, etc. You may use together.
  • an expression vector containing the expression cassette of the present invention containing the gene of interest can also be used as a vaccine or DNA vaccine for cancer or the like (Kaufman, HL et al., J. Clin. Oncol. 22, 2122-2132). (2004); McNeel, DG et al., J. Clin. Oncol. 27, 425-430 (2009)).
  • a cancer-specific antigen is used as a target protein, and an expression vector containing the expression cassette of the present invention incorporating a gene encoding the protein is administered into a living body by subcutaneous injection or the like, and the antigen is expressed in the living body. And activation of cellular immunity and antibody immunity of the host against the antigen can be induced.
  • the expression cassette of the present invention can be used for prevention and treatment of diseases such as cancer.
  • the present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
  • Construct No. of the expression cassette used 1 (Fig. 7) It is a gene expression plasmid carrying a CMV i promoter upstream of a gene to be expressed. Further, BGH polyA (bovine growth hormone polyA additional sequence) is linked downstream of the gene. That is, it is considered that the protein expression ability is equivalent to that of an expression plasmid using a CMV i promoter that is generally used.
  • Construct No. 2 (Fig. 8) Construct No. CMV enhancer is inserted immediately downstream of 1 BGH poly A.
  • Construct No. 3 (Fig. 9) Construct No.
  • Construct No. 4 (Fig. 10) Construct No. The CMV enhancer was inserted immediately downstream of the 3 BGH poly A.
  • Construct No. 5 (Fig. 11) Construct No. The RU5 ′ region is inserted immediately downstream of the 3 human ⁇ -actin promoter.
  • Construct No. 6 (Fig. 12) Construct No. A CMV enhancer was inserted immediately downstream of 5 BGH poly A.
  • Construct No. 7 (Fig. 13) Construct No. A CMV enhancer is inserted immediately upstream of the human ⁇ -actin promoter.
  • Construct No. 8 (Fig. 14) Construct No. A CMV enhancer was inserted immediately downstream of 7 BGH poly A.
  • Construct No. 7 in which the RU5 ′ region has a reverse base sequence.
  • Construct No. 10 (FIG. 16) Construct No. A CMV enhancer is inserted immediately downstream of 9 BGH poly A.
  • Construct No. 11 (Fig. 17) Construct No. A 4 ⁇ CMV enhancer (4 CMV enhancers) is inserted immediately upstream of the 5 human ⁇ -actin promoter.
  • Construct No. 12 (Fig. 18) Construct No. CMV enhancer is inserted immediately downstream of 11 BGH poly A.
  • Construct No. 13 (FIG. 19) It is a gene expression plasmid carrying a CAG promoter.
  • FIG. 22-1) show the entire base sequence of pDNR-1r Donor vector (a plasmid vector for cloning without promoter (Clontech, product number PT3616-5)).
  • FIG. 23 shows the entire base sequence of pIDT-SMART vector (plasma vector for cloning without promoter (IDT)).
  • FIG. 24 shows the nucleotide sequence of the CMV i promoter (hCMV + intron promoter) region. The region was created by artificial synthesis based on the known information of the base sequence of the CMV i promoter region.
  • FIG. 25 shows the base sequence of the BGH polyA (3 ⁇ stop + BGH polyA) region. The part of “TAATAAA” in the base sequence is a more important sequence in the BGH polyA sequence.
  • FIG. 26 shows the base sequence of the CMV enhancer region.
  • the sequence was prepared by artificial synthesis based on the known information of the base sequence of the CMV enhancer region.
  • FIG. 27 shows the base sequence of the human ⁇ -actin promoter region. The sequence was prepared by obtaining a plasmid for pDRIVE-h ⁇ Actin-RU5 ′ from InvivoGen, and PCR amplifying the nucleotide sequence of that portion.
  • FIG. 28 shows the base sequence of the RU5 ′ forward direction ⁇ HTLV Type 1 long terminal repeat R segment and part of the U5 sequence (R-U5 ′) ⁇ region.
  • the sequence was prepared by artificial synthesis based on the known information of the base sequence of the R segment of ⁇ HTLV Type 1 long terminal repeat and part of U5 sequence (R-U5 ′) ⁇ region.
  • FIG. 29 shows the base sequence of the RU5 ′ reverse direction ⁇ HTLV Type 1 long terminal repeat R segment and part of the U5 sequence (R-U5 ′) ⁇ region.
  • the sequence was prepared by artificial synthesis based on the known information of the base sequence of the R segment of ⁇ HTLV Type 1 long terminal repeat and part of U5 sequence (R-U5 ′) ⁇ region.
  • FIG. 30 shows the base sequence of the 4 ⁇ CMV enhancer region. The underlined portion of the base sequence in FIG.
  • FIG. 31 shows the base sequence of the CAG promoter region. This is the base sequence of a known CAG promoter region. Based on the pCAGGS plasmid, which is a gene expression vector loaded with the CAG promoter, provided by Oriental Yeast Co., Ltd., it was prepared by PCR amplification of the nucleotide sequence of that portion.
  • FIG. 32 shows the base sequence of the 2IRES insert region. It is a base sequence of the IRES control gene of FIG.5 and FIG.6 of postscript.
  • BiP-IRES region lowercase part enclosed in frame (1) in base sequence of FIG. 32
  • Myc-IRES region lowercase part enclosed in frame (2) in base sequence of FIG. 32
  • the base sequence of two normal human DNAs is connected in this order, and is a gene used as a control gene for inserted genes.
  • the sequence was prepared by artificial synthesis based on the known information of the base sequences of the BiP-IRES region and Myc-IRES region.
  • FIG. 33 shows the nucleotide sequence of the SV40ori-UAS-CMVi-RU 5 ′ region. Construct No.
  • the base sequence of the insertion part on the left side of the insertion gene (foreign gene) of 14 (best construct).
  • the sequence was prepared by artificial synthesis based on the known information of the base sequence of each region included in the part.
  • the part surrounded by the frame (1) shows the SV40 ori region
  • the part enclosed by the frame (2) shows the CMV i promoter region
  • the part enclosed by the frame (3) shows , RU5 ′ region.
  • FIG. 34 shows the base sequence of the 3 ⁇ stop-BGH-polyA-UAS-hTERT enhancer + SV40 enhancer + CMV enhancer region.
  • 14 is the base sequence of the insertion part on the right side (downstream side) of the insertion gene (foreign gene) of 14 (best construct).
  • the sequence was prepared by artificial synthesis based on the known information of the base sequence of each region included in the part.
  • the uppercase part surrounded by the frame (1) encloses the BGH polyA region
  • the lowercase part enclosed by the frame (2) encloses the hTERT enhancer region by the frame (3).
  • the uppercase part indicates the SV40 enhancer region
  • the lowercase part surrounded by the frame (4) indicates the CMV enhancer region.
  • 35-1 and 35-2 show the structure No. The full base sequence of 14 vectors is shown.
  • (1) is SV-40 ori region
  • (2) is CMV i promoter region
  • (3) is RU5 ′ region
  • (4) is BiP IRES region
  • (5) is Myc IRES region
  • (6) Indicates the hTERT enhancer region
  • (7) indicates the SV40 enhancer region.
  • (2) Foreign gene used (inserted gene) 6His-S100A11-HA Using the cDNA of human normal fibroblasts as a template, PCR was prepared using a primer with a 6His linker on the 5 'side and a primer with an HA linker on the 3' side.
  • GFP Green Fluorescent Protein
  • REIC / Dkk-3 full length Primers were set using the cDNA of human normal fibroblasts as a template, and PCR was performed. N78-REIC-6His Primers were set so that 6His was attached to the 3 ′ side of the full-length REIC / Dkk-3 gene obtained above, and PCR was performed.
  • Control gene This IRES control gene referred to here is obtained by linking known base sequences of two normal human DNAs of BiP-IRES gene and Myc-IRES gene in this order, and was prepared by artificial synthesis. This gene was designated as construct no.
  • CD133-6His Primers were set using the cDNA of human normal fibroblasts as a template, and PCR was performed.
  • LGR5-HA Primers were set using the cDNA of human normal fibroblasts as a template, and PCR was performed.
  • Telomerase-6His Primers were set using the cDNA of human normal fibroblasts as a template, and PCR was performed.
  • KLF16 Primers were set using the cDNA of human normal fibroblasts as a template, and PCR was performed.
  • the cells were washed twice using PBS (phosphate buffered saline), and lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF, 5 ⁇ g / ml leupeptin, 5 ⁇ g) / Ml aprotinin, 2 mM Na 3 VO 4 , 1 mM NaF, 10 mM ⁇ -GP), and the protein was extracted. After centrifugation, the amount of protein in each supernatant used in each experiment was adjusted to the same concentration and diluted with the same amount of 2 ⁇ SDS sample buffer.
  • lysis buffer 50 mM HEPES, pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF, 5 ⁇ g / ml leupeptin, 5 ⁇ g) / Ml
  • Anti-HA antibody Cell Signaling Technology
  • Anti-GFP antibody Cell Signaling Technology
  • Anti-GFP antibody Cell Signaling Technology
  • Anti-REIC antibody rabbit anti-human REIC / Dkk-3 polyclonal antibody
  • Anti-6His antibody MBL
  • Anti-KLF16 antibody Abcam
  • Each cell line of HEK293 (derived from human normal kidney), MCF7 (derived from human breast cancer), PC3 (derived from human prostate cancer), HeLa (derived from human uterine cancer), HepG2 (derived from human liver cancer) is ATCC (the American Type Culture Collection). (Rockville, MD). The medium used was as follows.
  • HEK293 DMEM high glucose medium (Invitrogen) MCF7: DMEM high glucose medium (Invitrogen) PC3: F12 medium (Invitrogen) HeLa: DMEM high glucose medium (Invitrogen) HepG2: DMEM high glucose medium (Invitrogen)
  • the cell line was grown in the above medium supplemented with 10% (v / v) fetal bovine serum, penicillin (100 IU / ml) and streptomycin (100 ⁇ g / ml) and incubated under 5% CO 2 conditions. The results are shown in FIGS.
  • FIG. 1 shows the expression of various foreign genes transfected into HEK293 cell line using FuGENE TM -HD for 36 hours. Construct No.
  • the protein expression ability of 1 was equivalent to that of an expression plasmid using a CMV i promoter that is generally used (sold). That is, compared with the protein expression ability of these expression plasmids, the construct No. It can be said that the protein expression power of 2 is remarkably strong. In addition, similar results were obtained for the following various genes. 2 can be said to surpass the currently widely used gene expression system in the expression of all kinds of genes.
  • S100A11 considered to be involved in cancer growth; intracytoplasmic-nuclear translocation protein GFP: fluorescent protein; cytoplasmic protein REIC / Dkk-3 (full length): tumor suppressor protein; secretory protein N78-REIC: Originally produced fragment protein (fragment peptide consisting of amino acids 1 to 78 of the amino acid sequence of REIC / Dkk-3 protein shown in SEQ ID NO: 2)
  • CD133 Marker of cancer stem cells; also expressed on cell surface
  • LGR5 Marker of stem cells of normal cells and cancer cells; Transmembrane protein Telomerase: Involved in cell senescence and immortalization; Intracytoplasmic protein KLF16: Involved in protein transcription; Inner protein FIG.
  • FIG. 2 shows the expression of the KLF gene transfected into various cell lines with FuGENE TM -HD for 36 hours.
  • the construct No. 2 protein expression KLF16 gene expression
  • FIG. 3 shows the expression of the KLF gene transfected into the HEK293 cell line for 36 hours using various transfection reagents. In any transfection reagent, no. In the case of 2, the most KLF16 protein is expressed by the WB method.
  • FIG. 1 is a construct containing a CMV i promoter that is generally available and commonly used.
  • 13 is a construct containing a CAG promoter which is also commonly used. That is, protein expression in an amount that significantly surpasses constructs that are currently widely used and used as plasmid constructs for gene expression all over the world (improvement of the efficiency of the entire gene expression process). Made possible by using 2.
  • FIG. 4 shows the expression when the HEK293 cell line was transfected for 36 hours using the full-length REIC gene and the gene FuGENE TM -HD encoding N78-REIC. The left panel shows the structure No. by WB method. In construct No. 14, construct no.
  • the right panel shows N78-REIC-6His which is a fragment gene. 14 shows that the same can be said for the left panel. That is, the most useful protein expression among the displayed plasmid constructs (useful) is the construct No. 14.
  • FIG. 5 shows construct No. in which DNA encoding N78-REIC was inserted. 14 shows growth suppression and cell death induction of human prostate cancer cell PC3 cell line by No. 14.
  • Cell viability assay PC3 cell lines were seeded in complete media at a density of 50,000 cells per well in 6-well plates. After 24 hours of incubation, the cells were transfected into a given plasmid using FuGENE TM -HD reagent in complete medium and incubated for a given number of days.
  • FIG. 6 shows construct No. in which the full-length REIC gene was inserted.
  • 14 shows growth inhibition of human prostate cancer cell PC3 cell line by 14;
  • Cell viability assay PC3 cell lines were seeded in complete media at a density of 50,000 cells per well in 6-well plates. After 24 hours of incubation, cells were transfected into a given plasmid using FuGENE TM -HD reagent in Hank's Balanced Salt Solutions and incubated for a given number of days. After the incubation, cell viability was measured using CellTiter 96 (registered trademark) Aquaone One Solution Cell Proliferation Assay (Promega).
  • Expression cassette containing SV40 promoter or hTERT promoter An expression cassette according to the present invention containing GFP (Green fluorescein protein) as a target gene (inserted gene) is prepared, expressed by transfection of Hela cells and observation of GFP expression was analyzed for strength. Transfections were performed using Lipofectamine 2000, and GFP expression was observed under a fluorescence microscope 48 hours later.
  • the construct of the expression cassette used was the construct No. used in Example 1. 14 (FIG. 20) and construct no. Construct No. 14 in which CMV i promoter of 14 was replaced with SV40 promoter. 15 (FIG. 36) and construct no. Construct No. 14 in which the CMVi promoter of 14 was replaced with the hTERT promoter. 16 (FIG. 37).
  • the GFP gene was prepared by setting primers using pEGFP-N2, which is a GFP expression vector manufactured by Clontech, as a template, and using it by inserting it into the target gene insertion part of each construct.
  • pEGFP-N2 is a GFP expression vector manufactured by Clontech
  • a commercially available GFP expression plasmid pEGFP-N1 (Clontech) was used.
  • Construct No. The base structure of a partial fragment consisting of the 15 expression vector SV40 ori + UAS + SV40 enh + intron A + RU5 ′ regions is shown in FIG. 38, and its base sequence is shown in SEQ ID NO: 19.
  • FIG. 39 The structure of a partial fragment consisting of the 16 expression vector SV40 ori + UAS + hTERT enh + intron A + RU5 ′ regions is shown in FIG. 39, and the base sequence is shown in SEQ ID NO: 20. Furthermore, FIG. 40 shows the base sequence of DNA encoding GFP linked with a restriction enzyme site (SEQ ID NO: 21).
  • the portions surrounded by the frames (1), (2), (3) and (4) represent SV40 ori, SV40 promoter, intron A and RU5 ′, respectively, and the base sequence of FIG.
  • the part enclosed by the frame of (1), (2), (3) and (4) in the inside shows SV40 ori, hTERT promoter, intron A and RU5 ′, respectively.
  • FIGS. 41-1 and 41-2 The full-length base sequence of the vector in which the GFP gene is inserted into 14 expression vectors is shown in FIGS. 41-1 and 41-2 (continuation of FIG. 41-1) (SEQ ID NO: 22).
  • the construction No. The full-length base sequences of vectors obtained by inserting the GFP gene into 15 expression vectors are shown in FIGS. 42-1 and 42-2 (continuation of FIG. 41-1) (SEQ ID NO: 23).
  • construct no. The full-length base sequence of the vector in which the GFP gene is inserted into 16 expression vectors is shown in FIGS. 43-1 and 43-2 (continuation of FIG. 43-1) (SEQ ID NO: 24). In the base sequences of FIGS.
  • the parts surrounded by the frames (1), (2), (3) and (4) are the CMVi promoter (P-CMViRU), GFP gene, Myc, respectively.
  • IRES, 3 enhancers (pA-3enh) are shown, and the portions surrounded by the frames (1), (2), (3) and (4) in the base sequence of FIG. 42 are SV40 promoter (P-SViRU), respectively.
  • GFP gene, Myc IRES, 3 enhancers (pA-3enh) and the portions surrounded by the frames (1), (2), (3) and (4) in the base sequence of FIG.
  • the hTERT promoter (P-TiRU), GFP gene, Myc IRES, and 3 enhancers (pA-3enh) are shown.
  • FIG. 15 is a plasmid vector advantageous for strong gene expression in an environment where SV40 protein is highly expressed.
  • Reference numeral 16 denotes a plasmid vector advantageous for strong gene expression in an environment where hTERT protein is highly expressed in cancer cells and the like.
  • the results are shown in FIG. 44A, B, C and D respectively show a commercially available GFP expression plasmid (pEGFP-N1), construct No. 14 GFP expression plasmid, construct no. 15 GFP expression plasmids, and construct no.
  • the results of 16 GFP expression plasmids are shown.
  • the upper panel in the figure shows a bright field.
  • DNA encoding human erythropoietin linked with a restriction enzyme site SEQ ID NO: 25.
  • pTracer registered trademark
  • pEF6 / Myc-His-A
  • DNA encoding human erythropoietin was inserted into the site of Xba1.
  • FIG. 20 An expression cassette of the present invention containing human IgG light chain and human IgG heavy chain as a target gene (inserted gene) was prepared, HEK293 cells were transfected, and the expressed protein was analyzed by Western blot. The transfection was performed using FuGENE TM -HD, and human IgG light chain and human IgG heavy chain in the cell extract and cell culture medium were detected by Western blotting after 24 hours.
  • the construct of the expression cassette used was the construct No. used in Example 1. 14 (FIG. 20), and DNA encoding human IgG light chain or human IgG heavy chain was inserted as a target gene (inserted gene).
  • FIG. 20 DNA encoding human IgG light chain or human IgG heavy chain was inserted as a target gene (inserted gene).
  • FIG. 47 shows the nucleotide sequences of DNAs encoding human IgG light chain (FIG. 47A) and human IgG heavy chain (FIG. 47B) linked with restriction enzyme sites (SEQ ID NO: 26 and SEQ ID NO: 27, respectively).
  • restriction enzyme sites SEQ ID NO: 26 and SEQ ID NO: 27, respectively.
  • commercially available expression plasmids pTracer (registered trademark) -EF / V5-His-A (Invitrogen) and pEF6 / Myc-His-A (Invitrogen) were used, and the restriction enzyme sites EcoR1- of each plasmid were used.
  • DNA encoding human IgG light chain or human IgG heavy chain was inserted into the Xba1 site.
  • FIG. 48A shows the result of human IgG light chain
  • FIG. 48B shows the result of human IgG heavy chain.
  • lane 1 was expressed using pTracer (registered trademark) -EF / V5-His-A
  • lane 2 was expressed using pEF6 / Myc-His-A
  • lane 3 was Construct No. What was expressed using 14 is shown.
  • REIC protein using an expression vector obtained by modifying a commercially available plasmid vector
  • An expression cassette according to the present invention containing the human REIC gene as a foreign gene (inserted gene) is prepared, and HEK293 cells are transfected, Analyzed by blot. The transfection was performed using FuGENE TM HD, and after 24 hours, the REIC protein in the cell extract was detected by Western blotting.
  • the expression cassette used was constructed by inserting a DNA encoding REIC into the Xba1-Kpn1 insertion site of a commercially available pShuttle plasmid vector (Clontech), and further inserting a Kpn1-EcoR1 downstream of the DNA encoding the REIC. In the construction No.
  • Construct No. 14 (FIG. 20) was constructed by inserting three enhancers (hTERT enhancer, SV40 enhancer and CMV enhancer) downstream of the expression gene of the expression plasmid in the form of BGH poly A + 3 enhancers. 17 (FIG. 49).
  • a vector constructed by inserting a DNA encoding REIC into the Xba1-Kpn1 insertion site of a commercially available pShuttle plasmid vector (Clontech) was used.
  • FIG. 50 shows a construct No. in which DNA encoding REIC is inserted.
  • the entire base sequence of 17 expression vectors is shown (SEQ ID NO: 28). In the base sequence of FIG.
  • the portions surrounded by the frames (1) and (2) indicate DNA encoding REIC and three enhancers (3xenh), respectively.
  • Western blotting was performed using an anti-REIC antibody, and a cell extract 24 hours after transfection (total protein amount was 10 ⁇ g) was used. The results are shown in FIG.
  • Lane 1 is a result of cell extract of cells not expressing a foreign protein
  • Lane 2 is a result of cell extract of cells transfected with a vector encoding REIC into a commercially available pShuttle vector
  • Lane 3 is a commercially available pShuttle vector
  • the results of cell extracts of cells transfected with an expression vector in which DNA encoding REIC is inserted and further three enhancers are inserted downstream of the DNA encoding REIC (downstream of BGH poly A) are shown. As shown in FIG. The strongest expression was observed when 17 was used.
  • This result shows that the gene expression can be enhanced by incorporating the three enhancer parts of this system (construct backbone of construct No. 14) downstream of the expression gene cassette of a commercially available plasmid such as pShuttle vector. Indicates. That is, it is shown that it is possible to enhance the expression of the target gene by inserting three enhancer regions into various gene constructs.
  • the expression cassette used was constructed by inserting a DNA encoding REIC into the Xba1-Kpn1 insertion site of a commercially available pShuttle plasmid vector (Clontech), and further inserting a Kpn1-EcoR1 downstream of the DNA encoding the REIC.
  • a construct constructed by inserting three enhancers hTERT enhancer, SV40 enhancer and CMV enhancer
  • hTERT enhancer, SV40 enhancer and CMV enhancer existing downstream of the expression gene of expression plasmid 14 (FIG. 20) in the form of BGH poly A + 3 enhancers.
  • a vector constructed by inserting a DNA encoding human c-myc into the Xba1-Kpn1 insertion site of a commercially available pShuttle plasmid vector (Clontech) was used.
  • 52 shows the base sequence of the inserted c-myc gene (SEQ ID NO: 29)
  • FIG. 14 shows the base sequence (SEQ ID NO: 30) of the region containing BGH poly A and three enhancers contained in No. 14.
  • Western blotting was performed using an anti-c-mycC antibody (Santa Cruz, Cat No .: sc-70469), and a cell extract 24 hours after transfection (total protein amount was 10 ⁇ g). The results are shown in FIG.
  • Lane 1 is the result of cell extract of cells transfected with a vector encoding REIC into a commercially available pShuttle vector.
  • the result of the cell extract of the cell transfected with the expression vector inserted downstream of the DNA encoding myc (downstream of BGH poly A) is shown.
  • an expression vector in which a DNA encoding c-myc is inserted into a commercially available pShuttle vector and three enhancers are inserted downstream of the DNA encoding c-myc (downstream of BGH poly A) is used. The strongest expression was observed. This result shows that the gene expression can be enhanced by incorporating the three enhancer parts of this system (construct backbone of construct No.
  • iPS cells are believed to be useful in various types of regenerative medicine, i.e., the enhancer moiety can be used as a means to enable stronger gene expression of so-called reprogramming genes such as c-myc and the expressed protein itself.
  • reprogramming genes such as c-myc and the expressed protein itself.
  • the expression cassette of the present invention containing GFP (Green fluorescein protein) as a foreign gene (inserted gene) was prepared, HEK293 cells were transfected, and the expressed protein was analyzed by Western blot . Transfections were performed using FuGENE TM -HD, and GFP in the cell culture medium was detected by Western blotting after 24 hours.
  • the construct of the expression cassette used was the construct No. used in Example 1. 2 (FIG. 8) and construct no. 18, 19, 20 and 21.
  • Construct No. The structures of 18, 19, 20 and 21 are shown in FIGS. 55, 56, 57 and 58, respectively.
  • Construct No. 18 is a general gene expression vector containing the hTERT promoter.
  • Construct No. 19 is a plasmid in which the RU5 sequence is inserted upstream of the expression gene of a general gene expression vector (construct No. 18) containing the hTERT promoter.
  • Construct No. 20 is a plasmid in which an hTERT enhancer sequence is inserted downstream of the BGH poly A sequence of a general gene expression vector (construct No. 18) containing an hTERT promoter.
  • 21 is a plasmid in which an RU5 ′ sequence is inserted upstream of an expression gene of a general gene expression vector (construct No. 18) containing an hTERT promoter, and an hTERT enhancer sequence is inserted downstream of a BGH poly A sequence.
  • a general gene expression vector construct No. 18
  • an hTERT enhancer sequence is inserted downstream of a BGH poly A sequence.
  • the portions surrounded by the frames (1), (2), (3), (4) and (5) are the hTERT core promoter and the minimal CMV promoter ( (Minimal CMV promoter), RU5 ′, GFP gene, BGH poly A, hTERT core promoter.
  • the result of expression is shown in FIG.
  • lanes 2 to 6 are the same as construct Nos. 2, no. 18, no. 19, no. 20 and no.
  • the result made to express using 21 is shown.
  • this system (the form of the construct in which the target expression gene is sandwiched between a promoter and an enhancer) is a promoter of a gene weakly capable of gene expression, such as the hTERT promoter that enables cancer-specific gene expression. , which are useful for significantly enhancing gene expression.
  • Expression construct No. when co-transfecting multiple expression vectors incorporating different foreign genes 2 (FIG. 20), a plasmid having a DNA encoding DsRed (red fluorescent protein), construct No. 21 (FIG. 58), a plasmid in which DNA encoding Yeast GST (Gratathione S transferase) is incorporated, construct No. Plasmids incorporating DNA encoding GFP into 21 (FIG. 58) were simultaneously transfected into cells and the expressed proteins were analyzed by Western blot. Transfections were performed using FuGENE TM -HD.
  • HEK293 cells Hela cells, PC3 cells, HepG2 cells, HCT116 cells, MCF7 cell cancer cell lines are used, and human-derived fibroblast OUMS-24 cells and human-derived keratinocyte cells as normal cell lines NHK cells were used.
  • Each protein in the cell extract was detected by Western blotting 48 hours after transfection.
  • a DNA encoding DsRed and a DNA encoding Yeast GST were prepared by artificial synthesis based on a known cDNA sequence. Western blotting is performed using anti-GFP antibody, anti-6His antibody (MBL, used for detection of Telomerase added with 6His), anti-tubulin antibody (Sigma), anti-DsRed antibody (Clontech) and anti-Yeast GST antibody.
  • the plasmid construct (construct No. 21) containing the hTERT promoter is useful for gene expression specific to only cancer cells in various cancer cells and normal cells. That is, in this case, the gene is strongly expressed only in cancer cells, and gene expression in normal cells can be suppressed.
  • a plasmid construct (construct No. 2) containing a CMV promoter gene expression is observed in both human cancer cells and normal cells, and gene expression specific to cancer cells cannot be achieved.
  • a plasmid containing the hTERT promoter is useful in that gene expression can be achieved specifically in cancer cells.
  • FreeStyle 293-F cells (Invitrogen) are seeded in a 125 mL flask at a concentration of 5 to 6 ⁇ 10 5 cells / mL, The culture was shaken overnight (125 rpm) using Freestyle 293 Expression 1 Media (Invitrogen) at 37 ° C. in the presence of 8% CO 2 .
  • each of SGE-EPO-His tag and pTracer®-EF-EPO-His were adjusted to a concentration of 1 ⁇ 10 6 cells / mL and 293-F cells seeded in 30 mL in a 125 mL flask.
  • the tag plasmid DNA was mixed with a gene introduction reagent: 293 Fectin (Invitrogen) for gene introduction. After transfection, the cells were cultured with shaking in the presence of 8% CO 2 at 37 ° C. for 4 days, and the culture supernatant was collected.
  • the protein amount of this purified EPO protein was quantified by the Bradford method, and the protein amount obtained during 1 L culture was calculated from the purified protein amount during 25 mL culture (FIGS. 64A and B).
  • human erythropoietin can produce about 8 times the amount of protein compared to the expression level in the pTracer (registered trademark) -EF vector when the SGE vector is used. An extremely high expression level was achieved.
  • the culture supernatant was collected. 18 ⁇ L of this culture supernatant was separated using SDS-PAGE, and a glycosylated REIC protein having a molecular weight of about 55 kDa was detected by CBB staining (FIG. 65).
  • the collected culture supernatant is concentrated from 520 mL to 35 mL by ultrafiltration, the solvent is replaced with 20 mM Hepes Buffer (pH 7.2) using Sephadex TM G25M column chromatography (GE Healthcare), and the REIC protein The containing fraction was collected.
  • the protein was adsorbed using anion exchange column chromatography (DEAE-Toyopearl (registered trademark) 650M, Tosoh), and then a linear concentration gradient of sodium chloride (0) in 20 mM Hepes Buffer (pH 7.2).
  • the peak fraction of REIC protein was confirmed under the condition that the sodium chloride concentration was about 0.35M.
  • Each peak fraction was analyzed by SDS-PAGE, and a fraction composed only of high-purity REIC protein was recovered (FIG. 66).
  • the amount of protein of this purified REIC protein was calculated from the absorbance (280 nm), and further converted from the amount of purified protein during 520 mL culture to calculate the amount of protein obtained during 1 L culture (FIG. 67).
  • a gene comprising a DNA construct comprising a gene to be expressed and a poly
  • a addition sequence downstream of the first promoter of the present invention, and further comprising an enhancer or a second promoter linked downstream of the DNA construct Cassettes for mass production are achieved by ultra-high expression of the target protein to be expressed by gene expression regardless of cell type, gene type or transfection reagent type.
  • a wide range of applications are possible for application as a therapeutic protein drug and for clinical treatment, testing and diagnosis using genes.

Abstract

La présente invention concerne un procédé qui utilise des promoteurs, des amplificateurs et analogues pour accroître l'expression des gènes, notamment des gènes étrangers. L'invention concerne également une cassette pour accroître l'expression génétique comportant des promoteurs, des amplificateurs et analogues. La cassette pour accroître l'expression génétique contient un gène qui doit être exprimé et une construction d'ADN ayant une séquence d'addition de poly(A), qui sont en aval d'un premier promoteur, et contient également un second promoteur ou un amplificateur, en aval de la construction d'ADN.
PCT/JP2010/071196 2009-11-19 2010-11-19 Système pour accroître l'expression génétique, et vecteur de support pour un tel système WO2011062298A1 (fr)

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US13/510,719 US9493776B2 (en) 2009-11-19 2010-11-19 System for increasing gene expression and vector comprising the system
JP2011541992A JP5697042B2 (ja) 2009-11-19 2010-11-19 遺伝子発現を上昇させるシステム及び該システムを保持したベクター
AU2010322723A AU2010322723B9 (en) 2009-11-19 2010-11-19 System for increasing gene expression and vector comprising the system
CA2781332A CA2781332C (fr) 2009-11-19 2010-11-19 Systeme pour accroitre l'expression genetique, et vecteur de support pour un tel systeme
EP20100831684 EP2508603B1 (fr) 2009-11-19 2010-11-19 Système pour accroître l'expression génétique, et vecteur de support pour un tel système
CN201080061897.9A CN102741405B (zh) 2009-11-19 2010-11-19 提高基因表达的系统和保持有该系统的载体
KR1020127015275A KR101752941B1 (ko) 2009-11-19 2010-11-19 유전자 발현을 상승시키는 시스템 및 이 시스템을 유지한 벡터
RU2012125253/10A RU2577971C2 (ru) 2009-11-19 2010-11-19 Система для увеличения экспрессии генов и вектор, содержащий указанную систему
IL219882A IL219882A (en) 2009-11-19 2012-05-17 A system for amplifying a vector gene expression containing the system

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AU2010322723B2 (en) 2014-09-25
RU2012125253A (ru) 2013-12-27
CA2781332A1 (fr) 2011-05-26
US20120309050A1 (en) 2012-12-06
IL219882A (en) 2016-09-29
KR101752941B1 (ko) 2017-07-03
KR20120099718A (ko) 2012-09-11
JPWO2011062298A1 (ja) 2013-04-11
EP2508603A1 (fr) 2012-10-10
RU2577971C2 (ru) 2016-03-20
AU2010322723A1 (en) 2012-06-21
JP5697042B2 (ja) 2015-04-08
EP2508603A4 (fr) 2013-05-01
CN102741405B (zh) 2015-03-04
CA2781332C (fr) 2018-09-04
AU2010322723B9 (en) 2015-03-05
US9493776B2 (en) 2016-11-15
IL219882A0 (en) 2012-07-31

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